Nucleic acids encoding recombinant protein a

ABSTRACT

Disclosed are new recombinant nucleic acids encoding protein A polypeptides and methods of using these nucleic acids.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/952,082, filed on Dec. 6, 2007, which claims the benefit of priorU.S. Provisional Application No. 60/873,191, filed on Dec. 6, 2006. Thedisclosures of the prior applications are considered part of (and areincorporated by reference in) the disclosure of this application.

TECHNICAL FIELD

This invention relates to novel nucleic acids that encode truncatedrecombinant protein A polypeptides, vectors, cells, and methods of use.

BACKGROUND

Staphylococcal Protein A (SPA) is a protein that is found in natureanchored to the outer membrane of the gram-positive Staphylococcusaureus bacterium, the organism which is commonly associated withmedically significant human “Staph” infections. The role of SPA in thelife cycle of S. aureus remains uncertain, but some studies havecorrelated the presence of SPA with pathogenicity of the organism.

Functionally, SPA is well known for its ability to tightly, butreversibly, bind to the constant region of an immunoglobulin molecule(IgG). This property has been widely exploited in the affinitypurification of antibodies for commercial uses. For example, SPA can bepurified from S. aureus and covalently bound to various forms of solidsupports to thus immobilize it to make an affinity chromatography resin.Crude preparations of antibodies can then be passed over such animmobilized SPA resin to bind and capture the commercially valuableantibody, while contaminating materials are washed away. The boundantibody may then be eluted in pure form by a simple adjustment of thepH.

SUMMARY

The invention is based, at least in part, on new recombinant nucleicacid sequences encoding truncated versions of protein A polypeptides(e.g., rSPA) that (i) include some portion (but not all) of the X-domainof native protein A, (ii) do not include a signal sequence and (iii)bind specifically to an Fc region of an IgG immunoglobulin. The newnucleic acids have the advantage of being suitable for use inefficiently expressing a truncated form of protein A polypeptides innon-pathogenic bacteria, especially E. coli, without being significantlydegraded within the bacteria. Thus, the nucleic acids described hereincan be used in laboratory and/or manufacturing practices that do notrequire a pathogenic S. aureus host for the production of protein Apolypeptides. The truncated rSPA that is encoded by said the new nucleicacid sequences has the useful advantage that it contains some portion ofthe X domain, which portion significantly improves its ability to beimmobilized for use as an affinity chromatography reagent. A means ofefficiently producing a form of rSPA that contains some portion of the Xdomain in E. coli or other non-pathogenic bacteria, has not previouslybeen disclosed.

In one aspect, the invention features isolated nucleic acid moleculesthat include a nucleic acid sequence encoding truncated Staphylococcusaureus protein A polypeptides. The protein A polypeptides have one ormore of the following features: (i) includes less than a complete nativeX domain; (ii) does not include a signal sequence (e.g., the nucleotidedoes not encode a signal sequence) or a heterologous N-terminalsequence; (iii) binds specifically to an Fc region of an IgGimmunoglobulin; (iv) is not substantially degraded when expressed in aheterologous host (e.g., a non-Staphylococcal host such as E. coli); and(v) includes only Staphylococcal polypeptide sequences. The codingsequence can be codon-optimized for expression in a non-pathogenicorganism (e.g., E. coli). In some embodiments, the nucleic acid includesa sequence at least 80% identical (e.g., at least 85%, 90%, 95%, 98%,99%, or 100% identical) to SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:22.The nucleic acid sequence can be operably linked to a bacterial ribosomebinding site, e.g., ACGCGTGGAGGATGATTAA (SEQ ID NO:3). In someembodiments, the protein A polypeptides bind to the Fc region of humanIgG1 with an affinity of 1000 nM or less (e.g., 500 nM or less, 200 nMor less, 50 nM or less, 20 nM or less, 10 nM or less, or 5 nM or less)in 0.02 M sodium phosphate, pH 7.0.

The invention also features isolated nucleic acid molecules that encodea polypeptide, which include one or more nucleic acid sequences encodingan S. aureus protein A Ig-binding domain and a portion of an S. aureusprotein A X-domain, wherein the nucleic acid sequence encoding theportion of the X-domain has a stop codon at position 379, 382, 385, 388,391, 394, 397, 400, 403, 406, or 409 of the X domain coding sequence. Insome embodiments, the one or more sequences encoding an Ig bindingdomain are wild-type. In other embodiments, the one or more sequenceencoding an Ig binding domain are codon-optimized. In some embodiments,the sequence encoding the X domain is “wild-type” except for the stopcodon. In other embodiments, the sequence encoding the X domain iscodon-optimized. In some embodiments, the polypeptide sequence containsonly amino acid sequences found in a native Staphylococcus derivedprotein A.

In another aspect, the invention features vectors that include any ofthe nucleic acid molecules described herein. The vectors can beexpression vectors, wherein the polypeptide-encoding nucleic acidsequences are operably linked to expression control sequences (e.g., apromoter, activator, or repressor). The invention also featuresbacterial cells, e.g., non-pathogenic bacterial cells (e.g., E. coli),that include the above vectors and bacterial cells that includepolypeptide-encoding nucleic acid sequences described herein operablylinked to an expression control sequence. In other embodiments, theinvention also features bacterial cells, e.g., non-pathogenic bacterialcells (e.g., E. coli) transformed with the above vectors, and theprogeny of such cells, wherein the cells express a truncated protein Aor a polypeptide that includes a protein A Ig-binding domain and aportion of a protein A X domain.

The invention also features E. coli cells that include an exogenousnucleic acid molecule that encodes a polypeptide that includes SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:17. In some embodiments,the nucleic acid sequence that encodes the polypeptide iscodon-optimized for expression in E. coli. In some embodiments, thenucleic acid sequence includes SEQ ID NO:1 or SEQ ID NO:22. In someembodiments, the protein A binds to the Fc region of human IgG1 (e.g.,with an affinity of 1000 nM or less (e.g., 500 nM or less, 200 nM orless, 50 nM or less, 20 nM or less, 10 nM or less, or 5 nM or less)) in0.02 M sodium phosphate, pH 7.0.

In other embodiments, the invention features methods of producingtruncated protein A polypeptides that include one or more protein AIg-binding domains and a portion of a protein A X domain. The methodsinclude culturing any of the cells described herein under conditionspermitting expression of the polypeptide. The methods can furtherinclude purifying the protein A polypeptide from the cytoplasm of thecell. In some embodiments, the protein A polypeptide is then immobilizedon a solid support material, e.g., cellulose, agarose, nylon, or silica.In some embodiments, the solid substrate is a porous bead, a coatedparticle, or a controlled pore glass. The invention also features solidsupport materials on which the protein A polypeptide has beenimmobilized.

The invention also features methods of purifying a protein A polypeptidethat includes an Fc region of an IgG immunoglobulin. The methods includecontacting the truncated protein A polypeptide-bound substrate made asdescribed herein with a solution that includes a protein that includesan Fc region of an IgG immunoglobulin; washing the substrate; andeluting bound a polypeptide that includes an Fc region of an IgGimmunoglobulin. The invention also features protein A polypeptides(e.g., proteins that include an Fc region of an IgG immunoglobulin)purified by the methods described herein or using solid supportmaterials described herein.

As used herein, “truncated protein A polypeptide” refers to a protein Apolypeptide that (i) includes some, but not all, of a native X-domain,and (ii) binds specifically to an Fc region of an IgG immunoglobulin.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic map of native protein A domains.

FIG. 2 is an amino acid sequence (SEQ ID NO:4) of native Staphylococcusaureus (strain 8325-4) protein A (Lofdahl et al., Proc. Natl. Acad. Sci.USA, 80:697-701, 1983). N-terminal underlined sequence represents S.aureus signal peptide. C-terminal underlined sequence represents theX-domain.

FIG. 3 is an example of a protein A amino acid sequence broken into thedesignated domains: IgG binding E domain (SEQ ID NO:9), IgG binding Ddomain (SEQ ID NO:10), IgG binding A domain (SEQ ID NO:11), IgG bindingB domain (SEQ ID NO:12), IgG binding C domain (SEQ ID NO:13), X domain(X domain 1)(SEQ ID NO:14) and example of portion of X domain used tomake recombinant protein shown in FIG. 6 (X domain 2)(SEQ ID NO:15).

FIG. 4 is an amino acid sequence (SEQ ID NO:7) of an exemplary truncatedprotein A lacking portions of the X domain as seen in FIG. 3 as boldedamino acids. The sequences underlined in SEQ ID NO:7 are repetitiveeight amino acid sequences (KPGKEDXX); SEQ ID NO:8).

FIG. 5 is a second example of an amino acid sequence (SEQ ID NO:6) of arecombinant S. aureus protein A polypeptide with a portion of the Xdomain.

FIG. 6 is a plasmid map of pREV2.1-rSPA containing genetic elements forexpression of the rSPA recombinant gene. Sequence landmarks are notedand include the β-glucuronidase promoter, ribosome binding site (RBS),multiple cloning site (MCS) and Trp terminator. The plasmid backbone isdefined as the ˜3900 by DNA sequence between the MluI and BamHIrestriction sites.

FIG. 7 is a partial nucleotide sequence (SEQ ID NO:16) of an E. coliexpression vector. The nucleotide sequence includes a portion of thevector backbone at 5′ and 3′ terminal sequences (italics), the promotersequence is underlined, and the start methionine and the terminationcodon are in bold.

FIG. 8 is a depiction of an immunoblot using antibodies that bindspecifically to recombinant protein A polypeptides produced in E. colicells. Lane 1: rPA50; Lane 2: vector control; Lane 3; clone 7a; Lane 4:clone 9a; Lane 5: clone 10a; Lane 6: clone 19a.

FIGS. 9A-B are graphs comparing the results of dynamic binding capacityexperiments using (i) truncated protein A polypeptide produced using anucleic acid described herein and (ii) PROSEP™ A chromatography media(Millipore) as a commercially available comparison.

FIG. 10 is an exemplary nucleotide sequence (SEQ ID NO:2) that encodes atruncated protein A polypeptide.

FIG. 11 is an amino acid sequence (SEQ ID NO:17) of an exemplarytruncated protein A polypeptide lacking a portion of the X domain.

FIG. 12 is an amino acid sequence (SEQ ID NO:5) of an exemplarytruncated protein A polypeptide lacking a portion of the X domain.

FIG. 13 is an exemplary nucleic acid sequence (SEQ ID NO:22) encoding atruncated protein A polypeptide.

DETAILED DESCRIPTION

Described herein are novel nucleic acids and methods for the expressionof truncated forms of protein A that include some portion, but less thanall, of the native X-domain, only polypeptide sequences found in nativeS. aureus protein A, and bind specifically to IgG immunoglobulin Fcregion. The truncated forms of protein A can be expressedcytoplasmically (e.g., without a signal peptide) and harvested from anon-pathogenic host, for example, non-pathogenic strains of E. coli,which are generally considered safer to handle and use than S. aureus.Furthermore, molecular biological and fermentation techniques for E.coli have been developed that permit high levels of truncated protein Aexpression and recovery.

Structure of Full Length Protein A Precursor

SPA is a cell surface protein that can be isolated from particularstrains of Staphylococcus aureus. The protein is able to bind free IgGand IgG-complexes. Membrane-bound protein A has been identified in thefollowing S. aureus strains: NCTC 8325-4 (Iordanescu and Surdeanu, J.Gen. Microbiol., 96:277-281, 1976), NCTC 8530, i.e., Cowanl or ATCC12598; and SA113 or ATCC 35556. A soluble form of protein A is expressedby S. aureus strain A676 (Lindmark et al., Eur. J. Biochem., 74:623-628,1977). The ATCC strains described herein, as well as other S. aureusstrains, are available from American Tissue Culture Collection(Bethesda, Md.).

The gene encoding the full length SPA precursor is known as spa.Nucleotide and protein sequences for spa are publicly available, e.g.,through GENBANK nucleotide database at Accession No. J01786 (completecoding sequence) and/or BX571856.1 (genomic sequence of clinical S.aureus strain that includes coding sequence for GENPEPT Accession No.CAG39140). See also, U.S. Pat. No. 5,151,350. In spite of the varioussequences available to the public, the inventors believe that the newnucleic acid sequences described herein have not been previouslyisolated, sequenced, or publicly described.

Structurally, the SPA protein consists of an amino-terminal signalpeptide followed by five highly homologous immunoglobulin bindingdomains and a so-called X domain (see FIG. 1). The signal peptidedirects the SPA protein for secretion through the membrane and isthereafter removed by proteolysis. The five immunoglobulin bindingdomains, named A through E, are arranged as E-D-A-B-C in most naturallyoccurring forms of the molecule. The X domain, which lies at the carboxyterminus and is believed to be involved in anchoring the SPA to andextending it from the outer membrane of the bacterium, consists of twostructurally distinct regions, the first of which comprises a series ofhighly repetitive blocks of octapeptide sequence (termed Xr) and thesecond of which is a hydrophobic region at the extreme C-terminus(termed Xc), which is thought to anchor the SPA molecule into the cellmembrane. The entire SPA molecule thus consists of seven distinctdomains that are structurally arranged as [S]-E-D-A-B-C-X.

A number of strains of S. aureus are known and the protein sequence ofthe SPA from several of these has been reported in the prior art. Acomparison of these SPA sequences reveals a significant amount ofgenetic variability from one strain to another, which can include pointmutations, domain deletions, repetitive sequence insertions, and geneticrearrangements. The effect of such differences on SPA function has notbeen well studied, although it appears that deletion of at least aportion of the Xc domain results in a form of SPA that is secreted intothe culture medium (Lindmark et al., Eur. J. Biochem., 74:623-628,1977).

The IgG Fc region-binding domains of S. aureus include highly repetitivesequences at the protein level and, to a lesser extent, at the nucleicacid sequence level. Strain 8325-4 produces protein A that includes fiveIgG-binding domains that are schematically represented in FIG. 1 asregions E, D, A, B, and C. These domains bind specifically to the Fcand/or Fab portion of IgG immunoglobulins to at least partly inactivatean S. aureus-infected host's antibodies. By binding to the Fc region ofimmunoglobulins, protein A inhibits binding of IgGs to complement and Fcreceptors on phagocytic cells, thus blocking complement activation andopsonization.

The X domain is a C-terminal region that contains (i) Xr, a repetitiveregion with approximately twelve repetitive eight amino acid sequencesand (ii) Xc, an approximately 80 to 95 amino acid constant region at theC-terminus of the protein. Each repetitive amino acid sequence generallyincludes a KPGKEDXX (SEQ ID NO:8) motif, wherein in some embodiments theXX dipeptide can be NN, GN, or NK. See e.g., Uhlen et al., J. Biol.Chem. 259:1695-1702, 1984, and underlined residues in FIG. 5. The Xdomain is involved in the targeting and anchoring protein A to the cellsurface of S. aureus.

Although the X domain is not involved in IgG binding, it may be usefulto retain a portion of the X domain (e.g., when expression protein Apolypeptide by recombinant means) for the purpose of improving theproperties of the rSPA in the preparation of an affinity chromatographyresin. For example, a portion of the X domain can serve as a “molecularstalk” to tether the IgG-binding regions of the polypeptide to a solidsubstrate. Moreover, a portion of the X domain can act to present theIgG-binding regions of the polypeptide at a distance out and away from asolid substrate to which it is tethered in order to better allowinteractions of the IgG-binding regions to Fc-containing polypeptides.Further, the inclusion of a portion of the X domain can potentiallyimprove folding and/or stability of the protein A molecule over thefolding and/or stability of the protein A molecule without the X domain.Finally, certain of the amino acid side chains, e.g., lysine, present inthe X domain can provide convenient reaction sites to enable efficientcovalent coupling to a solid support without compromising the functionalproperties of the IgG binding domains.

The signal peptide (SP) is an N-terminal extension present in proteinsdestined for export by the general (Sec-dependent) bacterial secretionsystem. SP mediates recognition of the nascent unfolded polypeptidechain by the Sec-dependent secretion apparatus, translocation throughthe cell membrane, and cleavage by the signal peptidase (reviewed by vanWely et al., FEMS Microbiol. Rev., 25:437-54, 2001). Secretion issometimes necessary to achieve stable polypeptide expression.Cytoplasmic expression of recombinant proteins may fail because oftoxicity of the protein, a requirement of the secretion process forproper folding of the protein, or instability of the protein in thecytoplasmic environment. Stable recombinant protein expression cansometimes achieved by enclosing the polypeptide sequence of interestwith flanking regions of heterologous amino acids.

While it may be desirable to express an rSPA that contains at least aportion of the X domain, no demonstration of such a protein beingproduced free of heterologous sequences has been reported. Attempts toproduce a recombinant protein A containing a portion of the X domain bysecretion in E. coli produced a protein product that was extensivelydegraded by endogenous proteases (Uhlen et al., J. Bacteriol.,159:713-719, 1984). Another challenge that has been noted in attemptingto express a full-length rSPA gene product in E. coli is that the Xcregion can be toxic to the cells (Warnes et al., Curr. Microbiol.,26:337-344, 1993). These findings have led at least some investigatorsto eliminate the X domain when expressing the rSPA gene in E. coli (see,e.g., Hellebust et al., J. Bacteriol. 172:5030-34, 1990). The newsequences and systems described herein provide for high levels ofexpression in E. coli of proteolytically stable forms of rSPA thatcontain a portion of the X domain. These X domain containing forms ofrSPA have particular utility in the creation of rSPA containing affinitychromatography resins.

Virulence of S. Aureus

The Center for Disease Control and World Health Organization classify S.aureus a Biosafety Level II or Group II infectious agent, respectively.These classifications are reserved for agents associated with humandisease and hazards of percutaneous injury, ingestion, and/or mucousmembrane exposure.

S. aureus is a major cause of hospital-acquired (nosocomial) infectionsassociated with surgical wounds and implanted medical devices. Thisbacterium can release enterotoxins responsible for food poisoning, andsuperantigens released by S. aureus can induce toxic shock syndrome. S.aureus also causes a variety of suppurative (pus-forming) infections andtoxinoses in humans, as well as skin lesions including boils, styes, andfurunculosis. S. aureus has also been found to co-infect subjects withpneumonia, mastitis, phlebitis, meningitis, urinary tract infections,and deep-seated infections, such as osteomyelitis and endocarditis.

S. aureus expresses a number of potential virulence factors: (1) surfaceproteins that promote colonization of host tissues; (2) invasins (e.g.,leukocidin, kinases, hyaluronidase) that promote bacterial spread intissues; (3) surface factors (e.g., capsule, protein A) that inhibitphagocytic engulfment; (4) biochemical properties that enhance theirsurvival in phagocytes (carotenoids, catalase production); (5)immunological disguises (protein A, coagulase, clotting factor); (6)membrane-damaging toxins that lyse eukaryotic cell membranes(hemolysins, leukotoxin, and leukocidin); (7) exotoxins that damage hosttissues or otherwise provoke symptoms of disease (staphylococcalenterotoxins (SE) A-G, toxic shock syndrome toxin (TSST), exfoliativetoxin (ET)); and (8) inherent and acquired resistance to antimicrobialagents.

Thus, the virulence level of S. aureus is more severe than that forBiosafety Level 1 or Group 1 organisms, such as laboratory andcommercial strains of E. coli. Biosafety Level 1 is reserved forwell-characterized organisms not known to cause disease in healthy adulthumans, and of minimal potential hazard to laboratory personnel and theenvironment.

Nucleic Acids Encoding Truncated Protein A Polypeptides

In one aspect, described herein are certain nucleic acids encoding atruncated protein A polypeptide that has one or more of the followingcharacteristics: (i) contains only sequences coding for SPA, i.e., doesnot contain heterologous sequences, (ii) includes some portion of, butless than all of, the complete native X domain, (iii) binds specificallyto an IgG immunoglobulin Fc region, and (iv) lacks a signal sequence.Exemplary nucleic acids include, but are not limited to, nucleic acidsencoding SEQ ID NO:1 and variants thereof that have been codon optimizedfor expression in a specific host such as E. coli.

An exemplary nucleic acid encoding a truncated S. aureus protein Apolypeptide is as follows.

(SEQ ID NO: 1) ATGGCGCAACACGATGAAGCTCAACAGAACGCTTTTTACCAGGTACTGAACATGCCGAACCTGAACGCGGATCAGCGCAACGGTTTCATCCAGAGCCTGAAAGACGACCCTTCTCAGTCCGCAAACGTTCTGGGCGAGGCTCAGAAACTGAACGACAGCCAGGCCCCAAAAGCAGATGCTCAGCAAAATAACTTCAACAAGGACCAGCAGAGCGCATTCTACGAAATCCTGAACATGCCAAATCTGAACGAAGCTCAACGCAACGGCTTCATTCAGTCTCTGAAAGACGATCCGTCCCAGTCCACTAACGTTCTGGGTGAAGCTAAGAAGCTGAACGAATCCCAGGCACCAAAAGCAGACAACAACTTCAACAAAGAGCAGCAGAACGCTTTCTATGAAATCTTGAACATGCCTAACCTGAATGAAGAACAGCGTAACGGCTTCATCCAGTCTCTGAAGGACGACCCTAGCCAGTCTGCTAACCTGCTGTCCGAAGCAAAAAAACTGAACGAGTCCCAGGCTCCAAAAGCGGATAACAAATTCAACAAGGAGCAGCAGAACGCATTCTACGAAATCCTGCACCTGCCGAACCTGAACGAAGAACAGCGTAACGGTTTCATCCAATCCCTGAAAGACGATCCTTCCCAGTCCGCAAATCTGCTGGCAGAAGCAAAGAAACTGAACGACGCACAGGCACCGAAGGCTGACAACAAGTTCAACAAAGAGCAGCAGAATGCCTTCTACGAGATTCTGCATCTGCCAAACCTGACTGAGGAGCAGCGCAACGGTTTCATTCAGTCCCTGAAGGACGACCCAAGCGTCAGCAAGGAAATCCTGGCTGAGGCGAAAAAACTGAACGATGCACAGGCTCCGAAGGAAGAAGACAACAATAAACCTGGTAAAGAAGATAATAATAAGCCTGGCAAGGAAGATAACAACAAGCCGGGCAAGGAGGACAACAATAAACCGGGCAAAGAGGATAATAACAAGCCTGGTAAGGAAGACAACAACAAACCAGGCAAAGAAGATGGCAACAAGCCGGGTAAGGAGGATAATAAAAAACCAGGCAAGGAAGACGGCAACAAACCTGGCAAGGAGGATAACAAAAAGCCAGGCAAGGAGGATGGTAATAAACCGGGCAAAGAAGACGGCAACAAGCCTGGTAAAGAAGACGGTAACGGTGTACACGTCGTTAAACCTGGTGACACCGTGAACGACATCGCTAAGGCTAATGGCACCACGGCAGACAAGATTGCAGCGGACAATAAA TAA

For both SEQ ID NO:1 and SEQ ID NO:22, the E domain is encoded bynucleotides 2-171; the D domain is encoded by nucleotides 172-354; the Adomain is encoded by nucleotides 355-528; the B domain is encoded bynucleotides 529-702; the C domain is encoded by nucleotides 703-876; andthe X domain is encoded by nucleotides 877-1272.

Certain genes can provide challenges for efficient expression byrecombinant means in heterologous hosts. Alteration of the codons nativeto the sequence can facilitate more robust expression of these proteins.Codon preferences for abundantly expressed proteins have been determinedin a number of species, and can provide guidelines for codonsubstitution. Synthesis of codon-optimized sequences can be achieved bysubstitution of codons in cloned sequences, e.g., by site-directedmutagenesis, or by construction of oligonucleotides corresponding to theoptimized sequence by chemical synthesis. See, e.g., Mirzabekov et al.,J. Biol. Chem., 274:28745-50, 1999.

The optimization should also include consideration of other factors suchas the efficiency with which the sequence can be synthesized in vitro(e.g., as oligonucleotide segments) and the presence of other featuresthat affect expression of the nucleic acid in a cell. For example,sequences that result in RNAs predicted to have a high degree ofsecondary structure should be avoided. AT- and GC-rich sequences thatinterfere with DNA synthesis should also be avoided. Other motifs thatcan be detrimental to expression include internal TATA boxes, chi-sites,ribosomal entry sites, prokaryotic inhibitory motifs, cryptic splicedonor and acceptor sites, and branch points. These features can beidentified manually or by computer software and they can be excludedfrom the optimized sequences.

Nucleic acids described herein include recombinant DNA and synthetic(e.g., chemically synthesized) DNA. Nucleic acids also includerecombinant RNAs, e.g., RNAs transcribed (in vitro or in vivo) from therecombinant DNA described herein, or synthetic (e.g., chemicallysynthesized) RNA.

Nucleic acids can be double-stranded or single-stranded. Wheresingle-stranded, the nucleic acid can be a sense strand or an antisensestrand. Nucleic acids can be synthesized using oligonucleotide analogsor derivatives (e.g., inosine or phosphorothioate nucleotides). Sucholigonucleotides can be used, for example, to prepare nucleic acids thathave increased resistance to nucleases.

The term “purified,” referring, e.g., to a polypeptide, denotes amolecule that is substantially free of cellular or viral material withwhich it is naturally associated or recombinantly expressed, or chemicalprecursors or other chemicals used for chemical synthesis.

Also described herein are variants of nucleic acids encoding truncatedrSPA molecules. Such variants code for IgG-binding, truncated versionsof protein A polypeptides that (i) include a portion of but less thanthe complete X domain of SPA, (ii) are suitable for expression in E.coli, and (iii) are substantially identical to SEQ ID NO:6 or SEQ IDNO:7. In some embodiments, the nucleic acids do not encode a signalsequence. A variant nucleic acid (e.g., a codon-optimized nucleic acid)encoding a truncated protein A molecule can be substantially identical,i.e., at least 75% identical, e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 99.5% identical, to SEQ ID NO:1 or SEQ IDNO:22. In certain embodiments, a truncated rSPA variant that is“substantially identical” to SEQ ID NO:6 or SEQ ID NO:7 is a polypeptidethat is at least 75% identical (e.g., at least about 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical) to a SEQ IDNO:6 or SEQ ID NO:7.

The determination of percent identity between two nucleotide orpolypeptide sequences can be accomplished using the BLAST 2.0 program,which is available to the public at ncbi.nlm.nih.gov/BLAST. Sequencecomparison is performed using an ungapped alignment and using thedefault parameters (gap existence cost of 11, per residue gap cost of 1,and a lambda ratio of 0.85). When polypeptide sequences are compared, aBLOSUM 62 matrix is used. The mathematical algorithm used in BLASTprograms is described in Altschul et al., 1997, Nucleic Acids Research,25:3389-3402.

Nucleic acid variants of a sequence that contains SEQ ID NO:1, SEQ IDNO:2, or SEQ ID NO:22 include nucleic acids with a substitution,variation, modification, replacement, deletion, and/or addition of oneor more nucleotides (e.g., 2, 3, 4, 5, 6, 8, 10, 12, 15, 20, 25, 30, 35,40, 50, 60, 70, 80, 90, or 100 nucleotides) from a sequence thatcontains SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:22. All of theaforementioned nucleic acid variants encode a recombinant truncatedpolypeptide that (i) is suitable for expression in a non-pathogenic,heterologous host cell, (ii) contains a portion of, but less than allof, the complete X-domain of SPA, and (iii) specifically binds to IgG.In particular, the term “variant” covers nucleotide sequences encodingpolypeptides that are capable of binding to IgG through introduction ofadditional S. aureus protein A derived polypeptide sequences, forexample, from additional strains of S. aureus.

Vectors, Plasmids, and Host Cells

Nucleic acids encoding a truncated rSPA polypeptide as described hereincan be operably linked to genetic constructs, e.g., vectors andplasmids. In some cases a nucleic acid described herein is operablylinked to a transcription and/or translation sequence in an expressionvector to enable expression of a truncated rSPA polypeptide. By“operably linked,” it is meant that a selected nucleic acid, e.g., acoding sequence, is positioned such that it has an effect on, e.g., islocated adjacent to, one or more sequence elements, e.g., a promoterand/or ribosome binding site, which directs transcription and/ortranslation of the sequence.

Some sequence elements can be controlled such that transcription and/ortranslation of the selected nucleic acid can be selectively induced.Exemplary sequence elements include inducible promoters such as tac, T7,P_(BAD) (araBAD), and β-D-glucuronidase (uidA) promoter-based vectors.Control of inducible promoters in E. coli can be enhanced by operablylinking the promoter to a repressor element such as the lac operonrepressor (lac^(R)). In the specific case of a repressor element,“operably linked” means that a selected promoter sequence is positionednear enough to the repressor element that the repressor inhibitstranscription from the promoter (under repressive conditions).

Typically, expression plasmids and vectors include a selectable marker(e.g., antibiotic resistance gene such as Tet^(R) or Amp^(R)).Selectable markers are useful for selecting host cell transformants thatcontain a vector or plasmid. Selectable markers can also be used tomaintain (e.g., at a high copy number) a vector or plasmid in a hostcell. Commonly used bacterial host plasmids include pUC series ofplasmids and commercially available vectors, e.g., pAT153, pBR,PBLUESCRIPT, pBS, pGEM, pCAT, pEX, pT7, pMSG, pXT, pEMBL. Anotherexemplary plasmid is pREV2.1.

Plasmids that include a nucleic acid described herein can be transfectedor transformed into host cells for expression of truncated rSPApolypeptides. Techniques for transfection and transformation are knownin the art, including calcium phosphatase transformation andelectroporation. In certain embodiments, transformed host cells includenon-pathogenic prokaryotes capable of highly expressing recombinantproteins. Exemplary prokaryotic host cells include laboratory and/orindustrial strains of E. coli cells, such as BL21 or K12-derived strains(e.g., C600, DH1α, DH5α, HB101, INV1, JM109, TB1, TG1, and X-1Blue).Such strains are available from the ATCC or from commercial vendors suchas BD Biosciences Clontech (Palo Alto, Calif.) and Stratagene (La Jolla,Calif.). For detailed descriptions of nucleic acid manipulationtechniques, see Ausubel et al., eds., Current Protocols in MolecularBiology, Wiley Interscience, 2006, and Sambrook and Russell, MolecularCloning: A Laboratory Manual, Cold Spring Harbor Press, NY, 2001.

Expression and Purification of Truncated Protein A Polypeptides

Host cells containing a nucleic acid encoding a truncated rSPA can begrown under conditions suitable for expression of said encoded truncatedrSPA. Host cells can be grown that constitutively express truncatedrSPA. In other systems, host cells are first grown under conditions thatinhibit expression of truncated rSPA and are later switched to mediathat induces expression of truncated rSPA, for example, by activating orderepressing promoter operably linked to the rSPA coding sequence.

In another exemplary system, a bacterial host cell includes the codingsequence for a truncated rSPA (operably linked to T7 promoter), a T7 RNApolymerase (operably linked to lac operon/lac promoter control region),and a lac repressor (lacI gene). The lac repressor can bind to the lacoperon and prevent bacterial RNA polymerase binding to the lac promoterregion, thereby inhibiting T7 polymerase expression. Bacterial hostcells can be cultured, e.g., in fermentation tanks When the host culturereaches a desired population density (e.g., population reachesexponential or “log” growth), isopropyl-beta-D-thiogalactopyranoside(IPTG) is added to the bacterial growth media. IPTG binds to andinactivates the lac repressor, thereby derepressing the lac operon/lacpromoter and allowing expression of T7 polymerase. T7 polymeraseexpression, in turn, can drive high level expression of truncated rSPA.

After host cells have been grown under conditions suitable forexpression of truncated rSPA, host cells are harvested and rSPA proteinis purified from other host cell material. Typically, host cells arelysed in the presence of protease inhibitors and truncated rSPA isseparated from cell debris, e.g., by low speed centrifugation. Furtherenrichment of rSPA material is optionally accomplished by serialcentrifugations and isolation of fractions containing rSPA.

In certain embodiments, purification of truncated rSPA includes bindingto purification media such as a resin or magnetic beads. In theseembodiments, purification media includes IgG, or fragments thereof, thatbind to protein A. IgG fragments that bind to protein A include Fc orFab fragments. In other embodiments, purification media includesnickel-nitrilotriacetic acid (Ni-NTA), maltose, glutathione, or anyother material that binds to a truncated rSPA fusion protein. Afterbinding of truncated rSPA to purification media, the purification mediais washed, e.g., with a salt buffer or water, and truncated rSPA iseluted from the purification media with an elution buffer. Elutionbuffer includes a composition that disrupts truncated rSPA binding tothe purification media. For example, elution buffers can include glycineto disrupt IgG-truncated protein A interactions, imidazole or urea todisrupt His-tag-Ni-NTA interactions, and/or glutathione to disruptGST-glutathione interactions. Truncated rSPA is recovered by batch orcolumn elution.

Eluted rSPA can be further purified using chromatography techniques,e.g., ion exchange chromatography, affinity chromatography, gelfiltration (or size exclusion) chromatography. In addition, purifiedtruncated rSPA can be concentrated by binding a solution of purifiedrSPA to purification media and subsequently eluting bound truncated rSPAin a smaller volume of elution buffer.

For detailed protein purification techniques, see Scopes, ProteinPurification: Principles and Practice, Springer Science, NY, 1994.

Substrates

Described herein are new methods of making useful resins and othersubstrates to which truncated rSPA can be attached. Generally, a nucleicacid described herein is used to express truncated rSPA, which ispurified, and subsequently attached to a substrate. Substrates caninclude organic and inorganic materials. Substrates can be manufacturedin useful forms such as microplates, fibers, beads, films, plates,particles, strands, gels, tubing, spheres, capillaries, pads, slices, orslides. Substrate material can include, for example, magnetic beads,porous beads (e.g., controlled pore glass beads), cellulose, agarose(e.g., SEPHAROSE™), coated particles, glass, nylon, nitrocellulose, andsilica.

In some embodiments, truncated rSPA is expressed in a non-pathogenichost from a nucleic acid described herein, the rSPA is purified fromhost material, and the rSPA is attached (e.g., covalently attached) to aporous substrate that is hydrophobic and/or protein absorptive. Suchsubstrates or supports include ion exchange packings and bioaffinitypackings

In certain embodiments, truncated rSPA is harvested and purified asdescribed herein from a non-pathogenic organism, and the rSPA isattached to a porous protein-adsorptive support having hydroxyl groupson its surface. Exemplary supports include porous metalloid oxides,porous metallic oxides, and porous mixed metallic oxides. Such materialsinclude silica, e.g., silica particles or silica gel, alumina, stannia,titania, zirconia, and the like. In some embodiments, the porous supporthas a particle size of about 0.5 to about 800 micrometers, e.g., about 5to about 60 micrometers, and a pore diameter of about 30 to about 300angstroms, e.g., about 60 angstroms.

1. Exemplary Substrates—Porous Silica (Including Controlled Pore Glass)

Porous silica, including controlled pore glass, has been described inU.S. Pat. Nos. 3,549,524 and 3,758,284 and is commercially availablefrom vendors such as Prime Synthesis, Inc. (Aston, Pa.) and Millipore(Billerica, Mass.). Porous silica supports may undergo varioustreatments prior to being attached to truncated protein A polypeptides.Generally, a silica support is derivatized to introduce reactivefunctional groups. The derivatized support is activated and then coupledto truncated rSPA produced by the methods described herein.

For example, silica supports can be derivatized using anarginine-containing linker as described in U.S. Pat. No. 5,260,373.Silica supports can be amino-derivatized by a silanization process,e.g., using aminosilanes such as γ-aminopropyltrimethoxysilane6-(aminohexylaminopropyl)trimethoxy silane,aminoundecyltrimethoxysilane, p-aminophenyltrimethoxysilane,4-aminobutyltrimethoxysilane, and(aminotheylaminoethyl)-phenyltrimethoxysilane. Dual zone silanizationcan be employed, e.g., as described in U.S. Pat. Nos. 4,773,994,4,778,600, 4,782,040, 4,950,634, and 4,950,635. Silica supports can alsobe amino-derivatized using o-dianisidine, e.g., as described in U.S.Pat. No. 3,983,000. Amino-derivatized supports can becarboxy-derivatized by a second reaction with, e.g., succinic anhydride,e.g., as described in U.S. Pat. No. 4,681,870. Amino-derivatizedsupports can also be treated with an aldehydes, e.g., gluteraldehyde, tointroduce reactive aldehyde groups, as described in U.S. Pat. Nos.3,983,000 and 4,681,870. Derivatized porous silica can also be obtainedcommercially from Prime Synthesis, Inc.

Derivatized porous silica can be activated and reacted and bound totruncated rSPA in aqueous solution. For example, aqueous peptidesolutions react directly with o-dianisidine and/or gluteraldehyde coatedsubstrates. In other examples, carbodiimide and rSPA are mixed withderivatized substrate, such that carbodiimide reacts with and attachesrSPA to the derivatized substrate, e.g., as described in U.S. Pat. No.4,681,870.

Other methods for attaching a peptide to porous silica can also be usedin the methods described.

2. Exemplary Substrates—Agarose

A variety of agarose substrates known in the art can also be used in themethods described herein. For example, agarose substrates (e.g.,cross-linked, beaded agarose) suitable for use in chromatography packingresin can be derivatized, activated, and linked to a truncated rSPAproduced according to the methods described herein.

Derivatized agarose can be manufactured using methods known in the art.For example, agarose can be derivatized and activated using anarginine-containing linker as described in U.S. Pat. No. 5,260,373.Activated and derivatized agarose products suitable for peptide linkingare also commercially available from manufacturers such as AmershamBiosciences (Piscataway, N.J.). These include N-hydroxysuccinimide(NHS)-activated SEPHAROSE™ 4 FAST FLOW designed for the covalentcoupling through the primary amine of a ligand, CNBr-activatedSEPHAROSE™ designed for the attachment of larger primary aminecontaining ligands under mild conditions, EAH Sepharose 4B designed forcoupling of small ligands containing free carboxyl groups via a 10-atomspacer arm using carbodiimide as the coupling, and ECH Sepharose 4B forcoupling small ligands containing free amino groups via a 9-atom spacerarm also using carbodiimide as the coupling reagent. Instructions forcoupling derivatized SEPHAROSE™ to peptides can be obtained from themanufacturer.

Generally, truncated rSPA can be coupled to derivatized agarosesubstrates by incubating rSPA and the activated substrate in an aqueoussolution. Coupling conditions can include salt buffers such as4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), sodiumcarbonate, sodium chloride, potassium phosphate and other salts.Carbodiimide can also be used as needed or desired.

Applications For Truncated rSPA

Nucleic acids described herein are useful for the cost effective,efficient, and less hazardous production of truncated rSPA innon-pathogenic hosts such as E. coli as compared to harvesting ofsimilar peptides from S. aureus. The rSPA produced by the nucleic acidsdescribed herein can be covalently linked to substrates with greaterefficiency than forms of rSPA that are lacking the X domain.

Truncated rSPA can be also used in a wide array of industries includingresearch, medical diagnostics, and the discovery and manufacture oftherapeutic biologics. Research applications include use as a reagent inimmunoprecipitation and antibody purification protocols. Truncated rSPAcan be used as a component in diagnostic tools that isolate or evaluateantibodies in an organism.

Truncated rSPA is particularly useful for the manufacture of affinitychromatography resins that are widely used for large-scale purificationof antibodies for human therapeutic use. In these applications, atruncated rSPA containing affinity chromatography resin is contactedwith a solution containing a therapeutic antibody as well as undesiredcontaminating materials to selectively bind the desired antibody to theimmobilized rSPA. The rSPA containing affinity chromatography resin withthe desired antibody tightly bound to it is first washed to remove thecontaminating materials, and then the antibody is eluted from theaffinity chromatography resin in purified form by, for example, the useof acidic or high salt elution buffers.

The commercial significance of the therapeutic antibody market isexpected to grow quickly in the near future. For example, the globalmarket for therapeutic monoclonal antibodies in 2002 was reportedlyabove $5 billion and has been projected to approximately triple in sizeby 2008 to nearly $17 billion. Reichert and Pavlou, Nature Reviews DrugDiscovery, 3:383-4, 2004. Servicing this market will be benefited bycost-effective tools for large scale, reliable purification ofmonoclonal antibodies.

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

EXAMPLES Materials and Methods

All enzymes used in the procedures described herein were purchased fromNew England BioLabs (Ipswich, Mass.). All DNA purification kits werepurchased from Qiagen, Inc (Valencia, Calif.). All agarose plates werepurchased from Teknova, Inc (Hollister, Calif.). The engineered rSPA-swas synthesized and supplied in the plasmid pJ5:G03257. All E. colihosts were lab strains with the exception of Subcloning Efficiency™ DH5αcells, which were purchased from Invitrogen, Inc (Carlsbad, Calif.).

Preparation of Plasmid DNA Stocks

All liquid cultures were grown overnight for 12-16 hours at 37° C.,shaking at 250 RPM in disposable plastic baffled-bottom flasks. Allplasmid DNA was isolated using the QIAGEN Plasmid Maxi kit according tomanufacturers directions.

The pREV2.1 vector described in WO 90/03984 was digested with HpaI andNruI endonucleases to release the β-glucuronidase signal sequence and asmall 3′ portion of the β-glucuronidase promoter. The vector was thenre-ligated and subsequently digested with MluI and BamHI. As describedbelow, the PCR amplified optimized protein A coding sequence wasdigested MluI and BamHI and cloned by ligation with T4 ligase intodigested pREV2.1 vector to yield the construct pREV2.1-rSPA. FIG. 8shows a (partial) DNA sequence of the construct, in which the vectorsequence is indicated by italics, the promoter sequence is underlined,and the start methionine and the termination codons are in bold.

The pREV2.1-rSPA plasmid served as a source of the pREV2.1 plasmidbackbone. A vial of PR13/pREV2.1-rSPA was thawed and used to inoculate100 mL of Miller LB media (BD Biosciences, San Jose, Calif.) with 34μg/mL chloramphenicol and 100 μg/mL ampicillin. A HMS174/pET12a glycerolstock was scratched and used to inoculate 100 mL of LB media containing100 μg/mL of ampicillin.

Preparation of Transformation Competent E. Coli Host Cells

Both PR13 and BL21(DE3) host E. coli (Table 1) were prepared accordingto an adapted CaCl₂ protocol (Elec. J. Biotech., 8:114-120). Briefly,cells were grown under selection to an OD₆₀₀ ˜0.25-0.4, then harvestedin 50 mL Oak Ridge tubes (Nalgene, Rochester, N.Y.). Pellets wereresuspended in one-half volume ice-cold TB solution (10 mM PIPES, 75 mMCaCl₂.2H₂O, 250 mM KCl, 55 mM MnCl₂.4H2O, pH 6.7 with KOH) and incubatedon ice for 25 minutes. Cells were centrifuged at 8,000 RPM for 1 minuteat 4° C., TB was decanted and pellet was resuspended in one-tenthoriginal volume ice-cold TB. Aliquots of 100 μL were snap frozen inliquid nitrogen and stored at −80° C.

TABLE 1 E. coli strains described herein Strain Description PR13 Strainof E. coli used for protein expression with pREV2.1 expression system(F− thr-1 leuB6(Am) lacY1 rna-19 LAMpnp-13 rpsL132(Str^(R)) malT1(LamR)xyl-7 mtlA2 thi-1) BLR(DE3) Strain of E. coli used with pET expressionsystem (E. coli B F− ompT hsdSB(rB− mB−)gal dcm (DE3)Δ(srlrecA)306::Tn10 (Tet^(R)) DH5α Strain of E. coli used primarily forplasmid maintenance (F− φ80lacZΔM15 Δ(lacZYA-argF)U169 recA1 endA1hsdR17(rk−, mk+) phoA supE44 thi-1 gyrA96 relA1 λ−)

Example 1 Recombinant DNA Construct for Expression in E. Coli

To manufacture truncated rSPA in a host that is less pathogenic than S.aureus, a DNA construct was engineered to express a truncated version ofstrain 8325-4 protein A in E. coli. The construct contained a truncated8325-4 protein A coding sequence including the E, D, A, B, C, and partof the X domains, but missing both the native N-terminal S. aureussignal sequence and a portion of the native C-terminal X domain. The DNAconstruct did not introduce coding sequences for heterologouspolypeptides not found in native SPA. The coding sequence wasfunctionally linked to an E. coli promoter and E. coli ribosome bindingsite. Restriction digestions and ligations were performed according tomanufacturer instructions. PCR amplifications were performed asdescribed below. Ligations were transformed into E. coli strain DH5α.DNA sequencing of one of a DH5α clone (18A) was performed under contractwith the Iowa State University Sequencing Facility (Ames, Iowa) usingthe following primers: IPA-1: 5′ AAA GCA GAT GCT CAG CAA (SEQ ID NO:18);IPA-2: 5′ GAT TTC CTT GCT GAC GCT T (SEQ ID NO:19); Anti-IPA-2: 5′ AAGCGT CAG CAA GGAAAT C (SEQ ID NO:20); and BG promoter-2: 5′ GAT CTA TATCAC GCT GTG G (SEQ ID NO:21).

Example 2 Expression of Truncated rSPA in E. Coli

The ability of four independent DH5α clones (labeled PA/pREV 7a, 9a,10a, and 18a) harboring the construct described in Example 1 to expressrecombinant truncated rSPA was evaluated by SDS-PAGE and WesternBlotting. Total cell lysates from E. coli were electrophoresed onSDS-PAGE gels. Samples were analyzed by SDS-PAGE as described above.

SDS-PAGE results were consistent with the predicted molecular mass of˜47 kDA for recombinant truncated rSPA encoded by PA/pREV (FIG. 8). Theresults indicate that the constructs described herein can be abundantlyexpressed in E. coli without substantial degradation.

Example 3 Functional Characterization of Truncated rSPA Recovered fromE. Coli

Truncated rSPA (SEQ ID NO: 7) was attached to a controlled glass poreresin (CPG-PA) and its functional characteristics were evaluated in anumber of tests. To make the rSPA resin, truncated rSPA (from clone 18ain Example 2) was harvested and purified. Truncated rSPA was fused tocontrol pore glass beads and functional characteristics were compared tothose of Millipore's PROSEP® A High Capacity protein A controlled poreglass resin (Catalog No. 113115824).

Static Binding Assay

A static polyclonal binding assay was performed by equilibrating resinwith phosphate buffered saline (PBS) buffer pH 7.2. Polyclonal human IgG(hIgG) was added and allowed to incubate at room temperature for 30minutes with end over end mixing. The resin was washed with PBS bufferpH 7.2. The hIgG protein was eluted with 0.2 M Glycine pH 2.0. Theamount of hIgG in the eluate was determined by measuring UV adsorbanceat 280 nm, and the binding capacity calculated. The assay was performedthree consecutive times, using the same glass-bound protein A samples todetermine the persistent binding capacity of each product after repeateduse.

Results of the static binding assay indicate that CPG-rSPA has a similarstatic binding capacity to that of the PROSEP® A product. Bindingcapacity was determined to be 36.9+0.2 mg IgG per ml of CPG-rSPA resincompared to 35.2+0.4 mg IgG per ml resin of PROSEP® A product in thefirst cycle. The results in Table 2 indicate that after threeconsecutive binding experiments, neither product suffered significantreduction of binding capacity.

TABLE 2 Result (mg IgG/ml resin) Sample Cycle 1 Cycle 2 Cycle 3 CPG-rSPA36.9 ± 0.2 36.2 ± 0.4 36.0 ± 0.6 PROSEP A 35.2 ± 0.4 35.4 ± 1.2 34.7 ±1.0

Protein A Leaching

A protein A ELISA kit (from Repligen) was used to quantify the amount ofprotein A that leached into the eluates used to determine the staticbinding capacity shown in Table 2. ELISAs were performed as indicated bythe manufacturer.

Results in Table 3 indicate that less protein A leached into the firstcycle eluate from CPG-rSPA than from the PROSEP® A product. In secondand third cycles, protein A leaching was comparable for both protein Aresins.

TABLE 3 Result (ng PA/mg hIgG) Sample Cycle 1 Cycle 2 Cycle 3 CPG-rSPA14.1 ± 3.8   10 ± 1.1 11.4 ± 4.7 PROSEP A 31.9 ± 8.4 16.3 ± 2.1 10.1 ±1.4

Capacity Following Cleaning and Regeneration Exposure Cycles

Binding capacity for CPG-rSPA and PROSEP A resins were evaluatedsubsequent to regeneration and cleaning Resins were washed with 0.3% HClpH 1.5 and then exposed for 1 hour to 6 M Guanidine. Guanidine wasremoved by washing resins with 0.3% HCl pH 1.5, followed by anincubation period of 1 hour in the HCL solution. Following cleaning,each resin was equilibrated with PBS and the static hIgG bindingcapacity was measured as described above in section 1 (Static BindingAssay).

Results in Table 4 show no meaningful decrease in the binding capacityof the CPG-rSPA following three repeated cycles of HCL and Guanidineexposure consistent with PROSEP® A HC results.

TABLE 4 Result (mg IgG/ml resin) Sample Pre-clean Cycle 1 Cycle 2 Cycle3 CPG-rSPA 35.2 ± 0.2 35.1 ± 0.2 32.9 ± 1.0  33.3 ± 0.8 PROSEP A 34.8 ±1.2 31.9 ± 0.4 35.4 ± 2.52 34.6 ± 0.8

Non-Specific Protein Adsorption Following Cleaning and Regeneration

Each resin was incubated with Chinese Hamster Ovary (CHO) K1 cellconditioned medium containing 5% FBS at room temperature for 30 minutes.The resin was washed with PBS and then eluted with glycine pH 2.0 andneutralized with Tris buffer. Eluates were analyzed by (i) SDS-PAGE andsilver staining the protein gels and (ii) a CHO host Protein ELISA(Cygnus Technologies).

SDS-PAGE showed several non-specific protein bands for both CPG-rSPA andthe PROSEP® A HC that were similar in molecular weight and intensity(Data not shown). ELISA assay was not able to quantify bound host CHOproteins, indicating that both resins bind less than less than 5 ng CHOProtein/mg hIgG, the limit of detection for the assay.

Dynamic Binding Capacity

Dynamic binding breakthrough curves were generated by subjectingCPG-rSPA and PROSEP A to flow velocities of 100, 300, 500, and 700cm/hr. A feed stream of 1.0 mg/ml polyclonal human IgG was used with aresin volume of 0.5 ml and a column bed height of 2.5 cm. Capacity isreported at 10% breakthrough.

Under the conditions tested, CPG-PA performed comparably to PROSEP® A HCat each flow velocity. See Table 5 and FIG. 10.

TABLE 5 Capacity at 10% BT (mg IgG/ml resin) Sample 100 cm/hr 300 cm/hr500 cm/hr 700 cm/hr CPG-rSPA 19.5 6.2 6.4 5.3 PROSEP A 20 6.9 6.9 5.3

The results of the functional comparative analysis described hereinindicate that recombinant truncated rSPA expressed in E. coli, whenattached to a controlled pore glass resin, performed at least as welland, in some cases better, than Millipore's PROSEP A product, whichincorporates an SPA ligand derived from native S. aureus.

Example 4 Functional Advantage of a Truncated X Domain

This example demonstrates the advantage of rSPA containing a truncated Xdomain compared to rSPA without an X domain on chromatography resinimmunoglobulin binding capacities. rSPA and a Protein A (TPA), whichcontains the five Immunoglobulin binding domains, but does not containany of the X domain, were immobilized onto SEPHAROSE™ 4 Fast Flow resin(Amersham). The protein immobilizations were performed using equivalentmolar concentrations of each protein A under identical conditions. Astatic polyclonal human IgG (hIgG) binding assay was performed aspreviously described. The rSPA immobilized product had a 14 to 22%greater hIgG capacity than the X domain deficient TPA (see Table 6).

TABLE 6 Protein A Concentration Static hIgG Binding Sample [uM] Capacity[g/L] TPA 50 15.5 + 0.5 rSPA 50 18.8 + 0.7 TPA 200 41.4 + 1.7 rSPA 20047.2 + 1.1

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. An isolated nucleic acid molecule comprising a nucleic acid sequenceencoding a truncated protein A polypeptide that (i) includes someportion of, but less than all of, a complete native X domain; (ii) lacksa signal sequence; and (iii) binds specifically to an Fc region of anIgG immunoglobulin.
 2. The isolated nucleic acid molecule of claim 1,wherein the coding sequence is codon-optimized for expression in anon-pathogenic organism.
 3. The isolated nucleic acid molecule of claim1, wherein the nucleic acid comprises a sequence at least 90% identicalto SEQ ID NO:1 or SEQ ID NO:2.
 4. The isolated nucleic acid molecule ofclaim 3, wherein the nucleic acid comprises a sequence at least 95%identical to SEQ ID NO:1 or SEQ ID NO:2.
 5. The isolated nucleic acidmolecule of claim 4, wherein the nucleic acid comprises SEQ ID NO:1 orSEQ ID NO:2.
 6. The isolated nucleic acid molecule of claim 1, whereinthe sequence encoding a truncated protein A polypeptide is operablylinked to a bacterial ribosome binding site.
 7. (canceled)
 8. Anexpression vector comprising the nucleic acid molecule of claim 1operably linked to an expression control sequence.
 9. A bacterial cellcomprising the vector of claim
 8. 10. (canceled)
 11. A bacterial celltransformed with the vector of claim 8, or a progeny of the cell,wherein the cell expresses a truncated protein A polypeptide. 12-13.(canceled)
 14. A method of producing a truncated protein A polypeptide,the method comprising culturing the cell of claim 9 under conditionspermitting expression of the polypeptide.
 15. The method of claim 14,further comprising purifying the truncated protein A polypeptide fromthe cytoplasm of the cell.
 16. A method of producing a truncated proteinA polypeptide-containing affinity chromatography resin, said methodcomprising performing the method of claim 15 and immobilizing thetruncated protein A polypeptide on a solid support material. 17-19.(canceled)
 20. A method of purifying a protein comprising an Fc regionof an IgG immunoglobulin, the method comprising contacting the protein Apolypeptide-containing affinity chromatography resin made according toclaim 16 with a solution comprising a protein comprising an Fc region ofan IgG immunoglobulin; washing the substrate; and eluting bound proteincomprising an Fc region of an IgG immunoglobulin.
 21. An E. coli cellcomprising an exogenous nucleic acid molecule that encodes a polypeptideconsisting of SEQ ID NO:7.
 22. The cell of claim 21, wherein the codingsequence is codon-optimized for expression in E. coli.
 23. An isolatednucleic acid molecule that encodes a polypeptide comprising one or morenucleic acid sequences encoding a S. aureus protein A Ig-binding domainand a portion of a S. aureus protein A X-domain, wherein the nucleicacid sequence encoding the portion of the X-domain has a stop codon atposition 379, 382, 385, 388, 391, 394, 397, 400, 403, 406, or 409 of theX domain coding sequence.
 24. The nucleic acid of claim 23, wherein theone or more sequences encoding an Ig binding domain are wild-type. 25.The nucleic acid of claim 23, wherein the one or more sequence encodingan Ig binding domain are codon-optimized.
 26. The nucleic acid of claim23, wherein the sequence encoding the X domain is wild-type except forthe stop codon.
 27. The nucleic acid of claim 23, wherein the sequenceencoding the X domain is codon-optimized.
 28. (canceled)